Hypophosphatasia (HPP) is the inborn-error-of-metabolism that features rickets or osteomalacia due to loss-of-function mutation within the tissue-nonspecific alkaline phosphatase (TNAP) gene. To-date, there is no established medical treatment for this condition. Our work has clearly shown that TNAP knockout mice (Akp2-/- mice) faithfully mimic the severe infantile form of HPP, and that their rickets/osteomalacia is caused by accumulation of extracellular levels of inorganic pyrophosphate (ePPi), one of TNAP's natural substrates and a potent calcification inhibitor. In turn, high ePPi leads to a secondary elevation of skeletal osteopontin (OPN), which functions as another potent calcification inhibitor. In turn, overexpression of TNAP in skeletal tissues leads to increases in bone mineral density and bone volume fraction, via a mechanism that involves reduction in ePPi levels and increased dephosphorylation of skeletal OPN. Epileptic seizures lead to the early demise of Akp2-/- mice and are caused by inadequate utilization of pyridoxal-5'-phosphate (PLP - a hydrophilic form of Vitamin B6), another natural substrate of TNAP. In a major breakthrough during this last funding period, we have shown that enzyme replacement therapy (EzRT) using a bone-targeted form of TNAP is able to completely prevent all the symptoms of HPP in the Akp2-/- mice, including the epileptic seizures and the severe skeletal and dental abnormalities characteristic of this excellent model of infantile HPP. Our findings represent the first successful use of EzRT for a heritable primary disease of the skeleton, and are a foundation for future therapeutic trials for human HPP. With that ultimate goal in mind, the present competitive renewal application focuses sharply on optimizing this EzRT strategy in TNAP-deficient mouse models. Our Specific Aims are: I) To test the hypothesis that EzRT not only prevents HPP in Akp2-/- mice but can also rescue Akp2-/- mice with advanced HPP disease. Additionally, we will compare the relative efficacy of administering bone-targeted versus soluble TNAP in Akp2-/- mice. II) To test the hypothesis that EzRT will also be efficacious to prevent and treat adult HPP. We will compare the phenotypic abnormalities of three different mouse strains, i.e., a bone-specific TNAP knockout model (Akp2flox/flox; Col1a1-Cre), a transgenic model of dominant HPP (Col1a1- TnapD361V) and a ENU-mutagenesis model of semi-dominant HPP (Akp2Hpp/Hpp) and evaluate the efficacy of EzRT on the most representative model. III) To test the hypothesis that pharmacological activators of TNAP's pyrophosphatase activity can be used to stimulate bone mineralization in vitro and in vivo. We will also assess the ability of these TNAP activators to stimulate in vitro the residual TNAP activity present in serum samples from genotyped HPP patients to identify what TNAP mutations might respond to such a pharmacological intervention. Our work will have an immediate impact on the clinical management of HPP patients, while providing further insights into the pathogenesis of this variable disease. The novel TNAP activators that we have discovered may prove of interest for the prevention and treatment of osteoporosis.